Over the last decade, epigenetic phenomena have claimed a central role in the research of cell regulatory processes. In addition, there is increasing evidence that epigenetic factors may play a significant role in various human diseases. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) are known to exist in two functional states: unmethylated or methylated at the 5-position of the pyrimidine ring (5mC). Cytosines followed by guanine (CpG dinucleotides) are the preferred targets for methylation. Recent studies of genomic DNA from the human brain, neurons and from mouse embryonic stem cells detected evidence that CG sequences also contain 5- hydroxymethylcytosine (hmC). As their interactions with cellular proteins are distinct, the 5- hydroxymethyl groups in DNA do not merely mimic 5-methyl groups, but likely play an independent role in yet unknown epigenetic regulation of various biological processes, such as embryonic development, brain function, and cancer progression. However, further studies of these intriguing phenomena are hampered by the lack of adequate analytical techniques. A myriad of methods have been developed over the past several years to investigate DNA methylation profiles across large DNA regions - chromosomes and even entire genomes. All such methods are binary - i.e., designed to distinguish only the two epigenetic states of cytosine: methylated versus unmodified. This project is dedicated to the development of new technologies that are capable of unequivocal mapping of hmC in mammalian genomes. Our strategy is based on utilization of known and newly discovered enzymatic transformations of the hydroxylmethyl group in hmC to achieve selective detection and capture of hmC-containing genomic fragments. Combining these novel approaches with DNA microarray analyses, we will perform epigenome-wide mapping of hmC in the human brain. Finally, we will perform nucleotide-resolution mapping of hmC at selected loci to unveil the intra- and inter- individual variation of this novel cytosine modification in DNA. PUBLIC HEALTH RELEVANCE: Changes in DNA methylation patterns may predispose individuals to various human diseases, including cancer, schizophrenia, autism, asthma, and diabetes. Research into the epigenetic misregulation in human disease is hampered by the lack of adequate laboratory techniques. Our new technologies for the analysis of the 6th base, namely 5-hydroxymethylcytosine, will be an incredibly valuable addition to the "tool box" of comprehensive genomic studies.